Synthesis of the Trypanosoma cruzi LPPG heptasaccharyl myo-inositol

Article abstract of DOI:10.1021/ja057339b

Synthesis of the heptasaccharyl myo-inositol found in Trypanosoma cruzi lipopeptidophosphoglycan was accomplished using a convergent assembly of three building blocks. The target compound is the first complete 2-aminoethyl phosphonic acid substituted glyc

Full text of DOI:10.1021/ja057339b

Published on Web 02/18/2006
Synthesis of the Trypanosoma cruzi LPPG Heptasaccharyl
myo-Inositol
Markus Hederos and Peter Konradsson*
Contribution from the IFM Chemistry, Linko¨ping UniVersity, SE-581 83 Linko¨ping, Sweden
Received October 27, 2005; E-mail: petko@ifm.liu.se
Abstract: Synthesis of the heptasaccharyl myo-inositol found in Trypanosoma cruzi lipopeptidophospho-
glycan was accomplished using a convergent assembly of three building blocks. The target compound is
the first complete 2-aminoethyl phosphonic acid substituted glycan related to the glycosylphosphatidylinositol
anchor family to be synthesized. The order of assembly enables synthesis of phosphoinositol oligosac-
charides related to other glycosylinositolphospholipids in Tr. cruzi, the protozoan parasite causing Chagas’
disease, which is endemic in South America.
Introduction
different glycan structures can be expressed. All contain the
same tetramannoside framework linked to a nonacylated glu-
Chagas disease, caused by the protozoan parasite Trypano-
soma cruzi, is a major public health problem in Central and
South America, and 18-20 million people are currently infected
leading to ∼21 000 deaths each year.1 At present, there is no
cure for this disease, and the available drugs are used selectively
due to their cost, significant side effects, and low antiparasitic
activity in the prevalent chronic form of the disease.2 The
parasite has a complex life cycle with the mammalian host and
insect vector. Hematophagous reduViid vectors, infesting the
houses of poor people, transmit the parasite to vertebrates by
leaving parasite-containing feces after taking a bloodmeal.
Disease transmission also occurs through the transfusion of
infected blood, and the intake has recently been shown, through
ingestion of crushed sugar cane.3 Thus, elimination of the insect
vectors from homes through spraying with insecticide, which
has successfully diminished the number of newly infected
people, is not sufficient for complete eradication of this disease,
and a demand of new treatments and diagnostic tests remains.
The recently reported genome sequencing of T. cruzi CL Brener
clone, a T. cruzi II strain, may be a key to determining novel
targets for treatments.4 The organism has a large genome, which
plausibly contributes to mechanisms to evade host immune
responses.
cosamine substituted on O-6 with the uncommon 2-aminoethyl
phosphonic acid (2-AEP) motif.6 The digalactofuranosyl motif
appears to be characteristic of T. cruzi II strains that predominate
in human infections, while other structures contain phospho-
rylated substituents on the O-6 of the subterminal Man, which
is a direct analogue of the glycosylphosphatidylinositol (GPI)
anchor that anchors many of the parasitic membrane proteins
considered essential for host cell invasion. The T. cruzi GIPLs
are bifunctional molecules that up- and down-regulate different
cell types of the host immune system, with the glycan
component influencing B cells.7
The development of our synthetic route opens a number of
possibilities for future work, including the development of
immunogenic glycoconjugates for possible therapy or the
production of serum or monoclonal antibodies for diagnostic
purposes and the means to probe the immune regulatory
properties of different GIPL structures in detail. In addition,
smaller synthetic structures can be used in studies of GIPL and
GPI biosynthesis, which may reveal novel targets for therapeutic
intervention.
Results and Discussion
The inositolphosphoglycan 1 contains structural motifs (Galf,
2-AEP, and myo-Ins-1-PO4), which require a well-planned
synthetic strategy. For example, the presence of 2-AEP was a
synthetic challenge since protocols to compounds with this motif
are restricted because of the low abundance of natural com-
pounds containing 2-AEP. The disconnection sites of target
molecule 1 were chosen on the basis of our experience in the
successful synthesis of the Leishmania LPG core oligosaccha-
Herein, we present the synthesis of the heptasaccharyl myo-
inositol 1 (Figure 1), the inositolphosphoglycan of the T. cruzi
glycoinositolphospholipid (GIPL) formerly known as the lipo-
peptidophosphoglycan (LPPG).5 This glycan, attached to a
ceramide lipid anchor, constitutes a major cell membrane
constituent in the proliferative epimastigote stage of T. cruzi
that is found in the insect vector. Characterization of the GIPLs
from a number of T. cruzi strains has revealed that a number of
(6) Carreira, J. C.; Jones, C.; Wait, R.; Previato, J. O.; Mendonc¸a-Previato, L.
Glycoconjugate J. 1996, 13, 955-966.
(7) (a) DosReis, G. A.; Pec¸anha, L. M. T.; Bellio, M.; Previato, J. O.;
Mendonc¸a-Previato, L. Microbes Infect. 2002, 4, 1007-1013. (b) Ropert,
C.; Gazzinelli, R. T. Curr. Opin. Microbiol. 2000, 3, 395-403. (c) Previato,
J. O.; Wait, R.; Jones, C.; DosReis, G. A.; Todeschini, A. R.; Heise, N.;
Mendonc¸a-Previato, L. AdV. Parasitol. 2004, 56, 1-41.
(1) WHO, The World Health Report, 2002, www.who.int/tdr/diseases/chagas.
(2) Urbina, J. A.; Docampo, R. Trends Parasitol. 2003, 19, 495-501.
(3) Health Protection Agency, CDR Weekly 2005, 15.
(4) El-Sayed et al. Science 2005, 309, 409-415.
(5) de Lederkremer, R. M.; Lima, C.; Ramirez, M. I.; Ferguson, M. A. J.;
Homans, S. W.; Thomas-Oates, J. J. Biol. Chem. 1991, 266, 23670-23675.
9
3414
J. AM. CHEM. SOC. 2006, 128, 3414-3419
10.1021/ja057339b CCC: $33.50 © 2006 American Chemical Society

Synthesis of T. cruzi LPPG Glycan
A R T I C L E S
Figure 1. Target molecule 1 and the building blocks 2-4.
ride.8 In addition, the preparation and utilization of compound
4 has recently been presented.9 The introduction of this building
block was as late as possible in the synthetic route since it exists
as a diastereomeric mixture of carbon-phosphonates and requires
the largest number of synthetic steps. Synthesis of building block
2 was accomplished by condensation of two Galf-(â1 f 3)-
Manp disaccharides. A hexasaccharide donor was planned to
be formed by condensation of 2 and 3, followed by glycosylation
with derivative 4 to give a fully protected octasaccharide. This
order of assembly would open synthetic routes to other relevant
glycans found in T. cruzi.
Synthesis of compound 2 started with the regioselective
glycosylation of diol 610 with penta-O-acetyl-â-D-galactofura-
nose (5),11 with the 2-O-acetyl participating group of the
galactofuranoside steering the reaction to the 1,2-trans glycosidic
linkage. The Galf-(â1 f 3)-Manp disaccharide 7 was obtained
in 70% yield using tin tetrachloride activation. The acetates in
7 were exchanged to benzyls to give the fully benzylated
compound 8 in 86% yield using first sodium methoxide in CH2-
Cl2 and methanol and then treating the syrup obtained with
benzylbromide, silver(I) oxide, and potassium iodide in DMF.
Conversion of 8 into a bromoglycoside by treatment with
bromine followed by glycosylation with 7 using silver trifluo-
romethanesulfonate gave the desired tetrasaccharide 9 in 60%
yield. Hydrolysis of the thioglycoside was accomplished using
tetrabutylammonium periodate, trifluoromethanesulfonic acid,
and water in acetonitrile.12 Treating the obtained reducible
tetrasaccharide with DBU and trichloroacetonitrile13 in CH2Cl2
gave R-trichloroacetimidate 2 in 70% yield for the two steps
(Scheme 1).
Synthesis of building block 3 was accomplished by treating
ethyl 2-O-benzoyl-3,4,6-tri-O-benzyl-R-D-mannopyranosyl-(1 f
6)-2,3,4-tri-O-benzyl-1-R-D-thio-mannopyranoside (10)9 with
sodium methoxide in CH2Cl2 and methanol (2:1) to give
derivative 314 in 90% yield. Logically, attempts were performed
to couple thioglycoside 9 with 3 after conversion of the former
into the corresponding bromosugar, but without success. Con-
trary, compounds 2 and 3 were assembled into the desired
hexasaccharide 11 in good yield using trimethylsilyl trifluo-
romethanesulfonate as the promoter in diethyl ether (Scheme
2). This successful glycosylation emphasizes the usefulness of
thioglycosides as temporary anomeric protecting groups since
they can be transformed into other donors when necessary, not
leading to a deadlock in longer synthetic routes.
Dimethyl(methylthio)sulfonium trifluoromethanesulfonate
(DMTST) activation of donor 11 in the glycosylation with
compound 49 gave the fully protected octasaccharide 12 in 71%
yield (Scheme 3). The characterization was complicated by the
fact that compound 12 was obtained as a diastereomeric mixture
where the diastereomers could not be separated. However, this
problem was to be overcome after the final deprotections due
to the loss of the chirality of the carbon-phosphorus atom in
the target compound.
(8) Ruda, K.; Lindberg, J.; Garegg, P. J.; Oscarson, S.; Konradsson, P. J. Am.
Chem. Soc. 2000, 122, 11067-11072.
(9) Hederos, M.; Konradsson, P. J. Org. Chem. 2005, 70, 7196-7207.
(10) Sarbajna, S.; Misra, A. K.; Roy, N. Synth. Commun. 1998, 28, 2559-
2570.
(12) Hiromi, U.; Wakiyama, Y.; Mukaiyama, T. Chem. Lett. 1998, 567-568.
(13) Schmidt, R. R. Angew. Chem., Int. Ed. Engl. 1986, 25, 212-235.
(14) Xue, J.; Shao, N.; Guo, Z. J. Org. Chem. 2003, 68, 4020-4029.
(11) Lerner, L. M. Carbohydr. Res. 1996, 282, 189-192.
9
J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006 3415

A R T I C L E S
Hederos and Konradsson
Scheme 1. Synthesis of Building Block 2a
a Conditions: (a) SnCl4, 4 Å molecular sieves, -50 °C f rt, CH2Cl2, (70%); (b) 1. NaOMe, MeOH/CH2Cl2 (1:2); 2. BnBr, KI, Ag2O, DMF, (86%); (c)
Br2, CH2Cl2; (d) AgOTf, 4 Å molecular sieves, CH2Cl2, -30 °C, (60%); (e) 1. n-Bu4NIO4, TfOH, H2O, acetonitrile; 2. Cl3CCN, DBU, CH2Cl2, (70%).
Scheme 2. Synthesis of the Hexasaccharide Donora
a Conditions: (a) NaOMe, CH2Cl2/MeOH (2:1) (90%); (b) TMSOTf, Et2O, 4 Å molecular sieves, (89%).
Scheme 3. Assembly of the Fully Protected IPG and Subsequent Deprotection to 1a
a Conditions: (a) 11, DMTST, 4 Å molecular sieves, Et2O, (71%); (b) 1. NaOMe, CH2Cl2/MeOH (2:1); 2. Na (s), NH3 (l); 3. 0.1 M HCl, (83%).
Deprotection of such a complex molecule as 12 is always
crucial, and most often there is a limited number of attempts
due to the usually small amounts of compounds. Moreover, the
order of protecting group removal can be crucial, and the
particular deprotection sequence used here was a result from
experience in our own laboratory. Deprotection of 12 started
9
3416 J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006

Synthesis of T. cruzi LPPG Glycan
A R T I C L E S
(m, 1H), 5.38 (d, 1H, J ) 1.4 Hz), 7.20-7.35 (m, 10H); 13C (75.4
MHz, CDCl3), δ 14.7, 20.4, 20.5, 20.6, 20.7, 24.8, 62.4, 68.4, 68.8,
with deacetylation using sodium methoxide in CH2Cl2 and
MeOH (2:1), followed by debenzylation with sodium in liquid
ammonia. Finally, the camphor and isopropylidene acetals were
removed using 0.1 M aqueous hydrochloric acid. Changing this
particular order has been shown leading to phosphate migration
and a complex mixture of products on similar structures.8 Target
compound 1 was obtained in 83% yield after gel filtration. NMR
data were in good agreement with reported values from the
characterization of the T. cruzi GIPL.6 The anomeric carbons
of the two galactofuranoside residues were observed at 106.1
and 105.8 ppm, respectively, which is in accordance with the
unusual upfield shift observed from synthetic oligosaccharides
constituting the Galf-(â1 f 3)-Manp motif (Scheme 3).15
68.9, 71.4, 73.3 (2C), 74.6, 76.0, 76.3, 80.0, 82.7, 83.5, 101.8 (JCH
)
175 Hz), 127.5-128.3, 138.1, 138.3, 169.6, 169.9, 170.3 (2C); HRMS
calcd. for C36H46O14S: [M + Na]+ 757.2506; Found: 757.2543.
Ethyl (2,3,5,6-tetra-O-Benzyl-â-D-galactofuranosyl)-(1 f 3)-2,4,6-
tri-O-benzyl-1-thio-R-D-mannopyranoside (8). To a solution of
compound 7 (500 mg, 0.680 mmol) in CH2Cl2/MeOH (2:1, 15 mL)
was added NaOMe (4 mg, 0.074 mmol). The solution was stirred for
1 h, and Dowex-H+ was added, the mixture was filtered, and the filtrate
was concentrated. The obtained syrup was dissolved in dry DMF (10
mL), and benzyl bromide (1.62 mL, 13.6 mmol), KI (1.69 g, 10.2
mmol), and Ag2O (2.36 g, 10.2 mmol) were added. The mixture was
stirred for 4 h, diluted with toluene, and washed with water. The organic
phase was washed with saturated aqueous NaHCO3, dried, filtered, and
concentrated followed by FC (toluene/EtOAc 19:1 f 9:1) to give
compound 8 (595 mg, 0.585 mmol, 86%) as a colorless syrup. Rf )
0.42 (toluene/EtOAc 9:1); [R]D ) +11 (c 1.2, CHCl3); IR νmax cm-1
3440, 3086, 3062, 3029, 2919, 2867, 1496, 1453, 1384, 1207, 1102,
1028, 737, 697, 619; NMR: 1H (300 MHz, CDCl3), δ 1.26 (t, 3H, J )
7.5 Hz), 2.51-2.71 (m, 2H), 3.59 (dd, 1H, J ) 4.1 9.9 Hz), 3.67-
3.83 (m, 4H), 3.91-4.08 (m, 4H), 4.12-4.28 (m, 4H), 4.36-4.52 (m,
8H), 4.57-4.75 (m, 4H), 4.91 (d, 1H, J ) 11.0 Hz), 5.24 (s, 1H), 5.45
(d, 1H, J ) 1.9 Hz), 7.13-7.38 (m, 35H); 13C (75.4 MHz, CDCl3), δ
14.8, 25.1, 69.1, 71.1, 71.2, 71.7 (2C), 71.9, 73.0, 73.1, 73.3, 74.0,
74.3, 74.6, 75.4, 76.4, 81.2, 81.7, 83.1, 88.1, 101.8 (JCH ) 172 Hz),
127.1-128.3, 137.5-138.6; HRMS calcd. for C63H68O10S: [M + Na]+
1039.4431; Found: 1039.4426.
Conclusion
The T. cruzi LPPG heptasaccharyl myo-inositol was synthe-
1
sized using a convergent building block strategy. The H, 13C,
and 31P NMR spectra of compound 1 support the assignment
of the compound isolated from the insect vector.6 The order of
assembly and earlier synthesis of the LPPG core structure9
demonstrates the generality of the concept developed with a
view to future syntheses of similar phosphoinositol oligosac-
charides found in other T. cruzi glycoconjugates.
Experimental Procedures
General methods. Organic extracts were dried over MgSO4, filtered,
and concentrated in vacuo at 40 °C. NMR spectra were recorded at 25
°C. 1H and 13C chemical shifts are given in ppm relative to TMS (δ )
0.00) and CDCl3 (δ ) 77.0) in CDCl3, respectively, and acetone in
Ethyl (2,3,5,6-tetra-O-Benzyl-â-D-galactofuranosyl)-(1 f 3)-2,4,6-
tri-O-benzyl-R-D-mannopyranosyl)-(1 f 2)-[(2,3,5,6-tetra-O-acetyl-
â-D-galactofuranosyl)-(1 f 3) ]-4,6-di-O-benzyl-1-thio-R-D-man-
nopyranoside (9). Thioglycoside 8 (775 mg, 0.762 mmol) dissolved
in CH2Cl2 (15 mL) was treated with Br2 (42 µL, 0.815 mmol) for 10
min, diluted with dry toluene, and concentrated. The crude bromosugar
and compound 7 (560 mg, 0.762 mmol) were dissolved in CH2Cl2 (20
mL), and 4 Å molecular sieves were added. The mixture was cooled
to -40 °C and stirred for 10 min, and AgOTf (392 mg, 1.524 mmol)
was added. The mixture was stirred for 30 min, diluted with CH2Cl2,
filtered through Celite, washed with saturated aqueous NaHCO3, dried,
filtered, and concentrated. FC (toluene/EtOAc 6:1 f 3:1) gave 9 (785
mg, 0.457 mmol, 60%) as a syrup. Rf ) 0.29 (toluene/EtOAc 6:1);
[R]D ) +6 (c 1.0, CHCl3); IR νmax cm-1 3440, 3087, 3060, 3028, 2923,
2865, 1745, 1627, 1494, 1452, 1369, 1224, 1098,1026, 736, 697;
NMR: 1H (300 MHz, CDCl3), δ 1.19 (t, 3H, J ) 7.4 Hz), 1.93 (s,
3H), 1.97 (s, 3H), 1.98 (s, 3H), 2.12 (s, 3H), 2.49 (q, 2H, J ) 7.4 Hz),
3.50-3.57 (m, 1H), 3.69-3.91 (m, 9H), 4.08-4.64 (m, 24H), 4.69-
4.76 (m, 2H), 4.89-4.99 (m, 3H), 5.01 (s, 1H), 5.05-5.07 (d, 1H, J )
5.5 Hz), 5.20 (s, 1H), 5.26 (d, 1H, J ) 1.6 Hz), 5.31-5.37 (m, 1H),
5.45 (s, 1H), 5.65 (s, 1H), 7.17-7.45 (m, 45H); 13C (75.4 MHz, CDCl3),
δ 14.9, 20.4 (2C), 20.5, 20.6, 25.5, 62.4, 68.9, 69.1, 69.8, 71.3, 71.9
(2C), 72.2, 72.5, 72.8, 73.1 (2C), 73.2, 73.9, 74.4, 74.5 (3C), 74.7,
74.9, 75.0, 76.2, 76.4, 76.7, 80.2, 81.8, 82.0, 83.0, 83.5, 88.0, 100.7
(JCH ) 171 Hz), 101.7 (JCH ) 175 Hz), 102.6 (JCH ) 173 Hz), 127.0-
128.2, 137.7, 137.8, 138.2-138.6, 169.6, 169.8 (2C), 170.2; HRMS
calcd. for C97H108O24S: [M + Na]+ 1711.6849; Found: 1711.6918.
1-O-(2,3,5,6-tetra-O-Benzyl-â-D-galactofuranosyl)-(1 f 3)-2,4,6-
tri-O-benzyl-R-D-mannopyranosyl)-(1 f 2)-[(2,3,5,6-tetra-O-acetyl-
â-D-galactofuranosyl)-(1 f 3)]-4,6-di-O-benzyl-1-thio-R-D-man-
nopyranosyl trichloroacetimidate (2). To a solution of derivative 9
(590 mg, 0.349 mmol), H2O (100 µL), and TfOH (6 µL, 0.070 mmol)
in acetonitrile (25 mL) at 0 °C was added Et4NIO4 (61 mg, 0.140 mmol).
The solution was allowed to reach room temperature during 2.5 h,
diluted with CH2Cl2, and washed sequentially with aqueous saturated
NaHCO3 and 10% aqueous Na2S2O3. The organic phase was dried,
filtered, and concentrated followed by FC (toluene/EtOAc 3:1 f 1:1)
to give 2,3,5,6-tetra-O-benzyl-â-D-galactofuranosyl)-(1 f 3)-2,4,6-tri-
1
D2O (13C: δ ) 31.5, H: δ ) 2.225); 31P, 85% H3PO4 (δ ) 0.00),
was used as an external reference. pH* values in D2O were calibrated
against H2O-buffer solutions. TLC was performed on Silica Gel F254
plates with detection by UV light (254 nm) and/or by charring with
AMC [ammonium molybdate 10 g, cerium(IV)sulfate 2 g, dissolved
in 10% H2SO4 (200 mL)] followed by heating at ∼250 °C. Silica gel
(0.040-0.063 mm) was used for flash chromatography (FC). IR spectra
were recorded as KBr pellets (solids) or films on CaF2 crystals (syrups).
Gel filtrations were performed using deoxygenated H2O containing 1%
n-butanol as the eluent. Mass spectra were recorded at Stockholm
University Proteomics Facility, Dept. of Analytical Chemistry, Stock-
holm University.
Ethyl (2,3,5,6-tetra-O-Acetyl-â-D-galactofuranosyl)-(1 f 3)-4,6-
di-O-benzyl-1-thio-R-D-mannopyranoside (7). Ethyl 4,6-di-O-benzyl-
1-thio-R-D-mannopyranoside (6)10 (2.00 g, 4.94 mmol) and penta-O-
acetyl-â-D-galactofuranose (5)11 (2.31 g, 5.93 mmol) were dissolved
in CH2Cl2 (40 mL), and 4 Å molecular sieves were added. After stirring
for 10 min, the temperature was lowered to -50 °C, and SnCl4 (0.81
mL, 6.92 mmol) was added. The mixture was allowed to attain room
temperature and after 10 h was diluted with CH2Cl2 and filtered through
Celite, washed with saturated aqueous NaHCO3, dried, filtered, and
concentrated. FC (toluene/EtOAc 3:1 f 1:1) gave 7 (2.54 g, 3.46 mmol,
70%) as a colorless oil. Rf ) 0.39 (toluene/EtOAc 1:1); [R]D ) +60 (c
1.0, CHCl3); IR νmax cm-1 3482, 3029, 2926, 2869, 1748, 1638, 1496,
1452, 1382, 1372, 1227, 1092, 966, 739, 699, 602; NMR: 1H (300
MHz, CDCl3), δ 1.28 (t, 3H, J ) 7.4 Hz), 1.96 (s, 3H), 1.97 (s, 3H),
2.11 (s, 3H), 2.12 (s, 3H), 2.49-2.71 (m, 2H), 3.65 (dd, 1H, J ) 1.9
10.9 Hz), 3.77 (dd, 1H, J ) 4.3 10.9 Hz), 3.90 (dd, 1H, J ) 9.1 9.1
Hz), 4.07 (dd, 1H, J ) 3.0 9.1 Hz), 4.12-4.17 (m, 4H), 4.28 (dd, 1H,
J ) 3.6 6.0 Hz), 4.48 (d, 1H, J ) 12.3 Hz), 4.51 (d, 1H, J ) 11.0 Hz),
4.63 (d, 1H, J ) 12.3 Hz), 4.82 (d, 1H, J ) 11.0 Hz), 5.01 (d, 1H,
J ) 2.2 Hz), 5.07 (dd, 1H, J ) 2.2 6.0 Hz), 5.19 (s, 1H), 5.25-5.30
(15) Gorin, P. A. J.; Barreto-Bergter, E. M.; da Cruz, F. S. Carbohydr. Res.
1981, 88, 177-188.
9
J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006 3417

A R T I C L E S
Hederos and Konradsson
O-benzyl-R-D-mannopyranosyl)-(1 f 2)-[(2,3,5,6-tetra-O-acetyl-â-D-
galactofuranosyl)-(1 f 3)]-4,6-di -O-benzyl-1-thio-R-D-mannopyranose.
[Rf ) 0.42 (toluene/EtOAc 2:1:); NMR 13C (75.4 MHz, CDCl3), δ 20.5
(2C), 20.6, 20.7, 62.5, 69.2, 69.3, 69.9, 71.4, 71.7 (2C), 71.9, 72.0,
72.5, 73.0, 73.1 (2C), 73.2, 73.3, 73.9, 74.2, 74.3, 74.6, 74.7 (2C),
75.1, 76.3, 76.5, 80.2, 81.7, 82.0, 83.1, 88.2, 93.1, 100.6, 102.0, 103.1,
127.2-129.0, 137.8-138.7, 169.7, 169.9, 170.0, 170.4.]. To a stirred
solution of this residue and trichloroacetonitrile (700 µL, 6.980 mmol)
in CH2Cl2 (20 mL) at 0 °C, DBU (8 µL, 0.052 mmol) was added. The
reaction mixture was stirred overnight, diluted with toluene, and
concentrated. FC (toluene/EtOAc 6:1 + 1% Et3N) gave 2 (444 mg,
0.244 mmol, 70%) as a colorless oil. Rf ) 0.32 (toluene/EtOAc 6:1);
[R]D ) -4 (c 1.0, CHCl3); IR νmax cm-1 3459, 3086, 3063, 3030, 2919,
2863, 1745, 1673, 1495, 1454, 183, 1371, 1225, 1098, 1050, 1026,
969, 796, 737, 697, 645, 602; NMR: 1H (300 MHz, CDCl3), δ 1.89
(s, 3H), 1.91, (s, 3H), 1.95 (s, 3H), 2.10 (s, 3H), 3.47-3.52 (m, 1H),
3.62-3.83 (m, 7H), 3.93-4.59 (m, 25H), 4.64-4.72 (m, 3H), 4.86-
4.97 (m, 3H), 5.00-5.04 (m, 2H), 5.21 (s, 1H), 5.27 (d, 1H, J ) 2.5
Hz), 5.28-5.34 (m, 1H), 5.41 (s, 1H), 6.51 (d, 1H, J ) 1.4 Hz), 7.13-
7.41 (m, 45H), 8.52 (s, 1H); 13C (75.4 MHz, CDCl3), δ 20.4 (2C),
20.5, 20.7, 62.3, 68.5, 69.0, 69.1, 71.4, 72.0, 72.1 (2C), 72.3, 72.5,
73.0, 73.1 (2C), 73.3, 74.0, 74.3, 74.5, 74.6 (2C), 74.7, 75.0, 75.1,
76.3 (2C), 80.1, 81.8, 82.1, 83.1, 88.2, 90.9, 96.6 (JCH ) 180 Hz),
100.7 (JCH ) 170 Hz), 101.7 (JCH ) 172 Hz), 103.1 (JCH ) 173 Hz),
127.2-128.3, 137.7-138.6, 159.9, 169.6, 169.8, 169.9, 170.2; HRMS
calcd. for C97H104Cl3NO25: [M + Na]+ 1810.5861; Found: 1810.5824.
(1 f 2)-3,4,6-tri-O-benzyl-â-D-mannopyranosyl)-(1 f 6)-2,3,4-tri-
O-benzyl-R-D-mannopyranosyl-(1 f 4)-2-azido-3-O-benzyl-6-O-(2-
benzyloxycarbonylamino-ethyl)-phosphonic Acid Benzyl Ester-2-
deoxy-R-D-glucopyranosyl-(1 f 6)-1-di-O-benzyl-phosphoryl-4,5-
O-isopropylidene-2,3-O-(D-1,7,7-tri-methyl[2,2,1]bicyclohept-2-ylidene)-
D-myo-inositol (12). To a solution of 11 (235 mg, 0.091 mmol), 49
(85 mg, 0.070 mmol), and 4 Å molecular sieves in Et2O (15 mL) under
argon atmosphere, DMTST (50 mg, 0.193 mmol) was added. The
mixture was stirred for 4 h when Et3N (250 µL) was added. The mixture
was stirred for 20 min, diluted with toluene, filtered through Celite,
and concentrated. FC (toluene/EtOAc 4:1 f 1:1) gave compound 12
(186 mg, 0.050 mmol, 71%) as a diastereomeric mixture. Rf ) 0.59
(toluene/EtOAc 2:1); [R]D ) +14 (c 1.4, CHCl3); IR νmax cm-1 3430,
3088, 3061, 3030, 2925, 2857, 2105, 1745, 1727, 1498, 1455, 1372,
1228, 1115, 1050, 1026, 738, 698; NMR: 1H (600 MHz, CDCl3,
diastereomeric mixture), δ 0.88 (s, 6H), 0.94 (s, 6H), 1.03 (s, 3H),
1.04 (s, 3H), 1.20-1.24 (m, 2H), 1.37-1.47 (m, 16H), 1.70-1.76 (m,
4H), 1.86-2.08 (m, 32H), 3.27-5.13 (m, 182H), 5.18 (s, 1H), 5.20-
5.22 (m, 2H), 5.24 (s, 1H), 5.26 (s, 1H), 5.29-5.32 (m, 3H), 5.37 (s,
1H), 5.40 (s, 1H), 5.46 (s, 1H), 5.47 (s, 1H), 5.91 (m, 1H), 6.02 (m,
1H), 7.05-7.47 (m, 200H); 13C (150.9 MHz, CDCl3, diastereomeric
mixture), δ 9.9 (2C), 20.1 (2C), 20.3 (5C), 20.4, 20.5 (3C), 20.6, 26.2
(J ) 139 Hz), 26.4 (J ) 139 Hz), 26.8 (3C), 26.9, 27.0 (2C), 29.7
(2C), 35.0 (2C), 43.7 (2C), 44.9 (2C), 47.8 (2C), 51.4 (2C), 62.3 (2C),
62.5, 62.6, 63.6, 64.1, 66.1 (3C), 66.2, 66.9 (J ) 5.9 Hz), 67.1 (J )
5.9 Hz), 68.7 (2C), 68.8, 68.9 (2C), 69.2, 69.3 (2C), 69.4-69.5 (several
C), 69.6 (2C), 70.7, 70.9, 71.2 (2C), 71.8 (2C), 71.9 (2C), 72.0-72.2
(several C), 72.3, 72.4, 72.7-72.8 (several C), 72.9 (2C), 73.0 (2C),
73.2 (2C), 73.5, 73.6 (2C), 73.8 (2C), 74.0, 74.1, 74.3, 74.4-74.9
(several C), 75.0, 75.2, 75.4, 75.7 (2C), 76.2 (2C), 76.4 (2C), 76.6
(2C), 77.2-77.6 (several C), 78.4, 78.7, 79.7, 79.8, 80.3, 80.4, 81.7,
81.8 (2C), 81.8, 82.9, 83.0, 87.9 (2C), 96.5, 96.6, 98.9, 99.0, 99.7,
99.9, 100.0, 100.3, 100.4, 100.5, 102.1, 102.4, 102.8 (2C), 112.5 (2C),
119.0 (2C), 126.6-128.6, 135.3-136.6, 137.4-138.8, 156.0 (2C),
169.4 (2C), 169.8, 169.9, 170.0 (4C); 31P (decoupled, 121 MHz, CDCl3,
diastereomeric mixture), δ -0.32, -0.16, 31.6, 32.3; HRMS calcd.
for C212H234N4O51P2: [M + 2Na]2+ 1879.7555; Found: 1879.7485.
Ethyl (3,4,6-tri-O-Benzyl-â-D-mannopyranosyl)-(1 f 6)-2,3,4-tri-
O-benzyl-1-thio-R-D-mannopyranoside (3). To compound 109 (400
mg, 0.388 mmol) dissolved in CH2Cl2/MeOH (2:1, 10 mL), NaOMe
(20 mg, 0.370 mmol) was added. The solution was stirred overnight,
Dowex-H+ was added, the solution was filtered, and the filtrate was
concentrated. FC (toluene/EtOAc 3:1) gave 3 (324 mg, 0.349 mmol,
90%) as a colorless syrup. Rf ) 0.49 (toluene/EtOAc 2:1:); [R]D
)
+82 (c 1.0, CHCl3); IR νmax cm-1 3467, 3088, 3063, 3029, 2925, 2869,
1496, 1452, 1384, 1363, 1209, 1100, 1050, 1027, 911, 845, 789, 736,
697; NMR spectra were in accordance with those previously pub-
lished.14
Ethyl (2,3,5,6-tetra-O-Benzyl-â-D-galactofuranosyl)-(1 f 3)-2,4,6-
tri-O-benzyl-R-D-mannopyranosyl)-(1 f 2)-[(2,3,5,6-tetra-O-acetyl-
â-D-galactofuranosyl)-(1 f 3) ]-4,6-di-O-benzyl-R-D-mannopyranosyl-
(1 f 2)-3,4,6-tri-O-benzyl-â-D-mannopyranosyl)-(1 f 6)-2,3,4-tri-
O-benzyl-1-thio-R-D-mannopyranoside (11). Compounds 2 (360 mg,
0.198 mmol) and 3 (257 mg, 0.277 mmol) were dissolved in Et2O (20
mL), and 4 Å molecular sieves were added. The mixture was cooled
at 0 °C for 30 min, and TMSOTf (8 µL, 0.040 mmol) was added. After
1 h, solid NaHCO3 was added, and the mixture was filtered through
Celite and concentrated. FC (toluene/EtOAc 6:1) gave hexasaccharide
11 (455 mg, 0.176 mmol, 89%) as a syrup. Rf ) 0.41 (toluene/EtOAc
6:1); [R]D ) +13 (c 0.9, CHCl3); IR νmax cm-1 3463, 3089, 3064, 3031,
2916, 2861, 1747, 1634, 1497, 1454, 1369, 1226, 1099, 1076, 1050,
1028, 738, 699; NMR: 1H (300 MHz, CDCl3), δ 1.14 (t, 3H, J ) 7.4
Hz), 1.88 (s, 3H), 1.92 (s, 3H), 1.94 (s, 3H), 2.05 (s, 3H), 2.35-2.57
(m, 2H), 3.49-4.75 (m, 57H), 4.80-4.89 (m, 4H), 4.94-4.98 (m, 2H),
5.02-5.05 (m, 3H), 5.21 (s, 2H), 5.26-5.32 (m, 2H), 5.37 (s, 1H),
5.43 (s, 1H), 7.09-7.44 (m, 75H); 13C (75.4 MHz, CDCl3), δ 14.8,
20.3, 20.4, 20.5, 20.6, 25.0, 62.3, 66.5, 69.0, 69.1, 69.2, 69.4, 71.0,
71.2, 71.3, 71.5, 71.8-71.9 (several C), 72.5, 72.7, 72.8, 73.1, 73.2,
73.8, 74.2, 74.5, 74.7-74.8 (several C), 75.0, 75.5, 76.2, 76.3, 76.5,
76.5, 78.9, 80.3, 80.5, 81.4, 81.8, 82.0, 83.1, 88.1, 98.5 (JCH ) 172
Hz), 100.4 (JCH ) 173 Hz), 100.7 (JCH ) 173 Hz), 102.1 (JCH ) 176
Hz), 102.6 (JCH ) 175 Hz), 127.1-128.9, 137.7-138.8, 169.4, 169.8,
169.9, 170.0; HRMS calcd. for C151H164O34S: [M + Na]+ 2576.0722;
Found: 2576.0781.
(â-D-Galactofuranosyl)-(1 f 3)-(r-D-mannopyranosyl)-(1 f 2)-
[(â-D-galactofuranosyl)-(1 f 3)]-r-D-mannopyranosyl-(1 f 2)-â-
D-mannopyranosyl)-(1 f 6 )-r-D-mannopyranosyl-(1 f 4)-2-amino-
6-O-(2-amino-ethyl)-phosphonic Acid 2-Deoxy-r-D-glucopyranosyl-
(1 f 6)-1-O-phosphate-D-myo-inositol, Ammonium Salt (1). To a
solution of diastereomeric 12 (105 mg, 0.0279 mmol) in CH2Cl2/MeOH
(6 mL, 2:1) was added NaOMe (2 mg, 0.0370 mmol). The solution
was stirred for 1 h, diluted with CH2Cl2, and washed with 0.5 M
aqueous NH4Cl. The organic phase was dried, filtered, and concentrated.
The obtained residue was dissolved in dry THF (∼2 mL) and added to
∼20 mL of NH3 (l) at -33 °C. A small piece of sodium was added,
turning the mixture to dark blue. Stirring was continued for 1 min when
NH4Cl (s) was added until the blue color disappeared. The mixture
was concentrated under a stream of N2 (g), and 10 mL of 0.1 M aqueous
HCl was added. The mixture was stirred for 5 h and neutralized with
0.1 mL of NH3. The aqueous phase was washed with Et2O (15 mL)
and concentrated. The obtained residue was purified on a Sephadex
G-15 column using H2O containing 1% n-butanol to give compound 1
(35.2 mg, 0.0231 mmol, 83%) as a solid. [R]D ) +28 (c 1.4, H2O);
NMR: 1H (600 MHz, D2O), δ 1.97-2.14 (m, 2H), 3.18-3.28 (m, 2H),
3.87-3.43 (m, 2H), 3.57 (dd, 1H, J ) 3.0 10.2 Hz), 3.64-3.86 (m,
21H), 3.91-3.95 (m, 6H), 3.98-4.00 (m, 1H), 4.03-4.16 (m, 13H),
4.19 (m, 1H), 4.25-4.28 (m, 2H), 4.35 (m, 1H), 5.13 (s, 1H), 5.14 (s,
1H), 5.17 (s, 1H), 5.20 (s, 1H), 5.29 (s, 1H), 5.31 (s, 1H), 5.66 (d, 1H,
J ) 3.6 Hz); 13C (150.9 MHz, D2O), δ 25.6 (J ) 134 Hz), 36.6, 55.0,
66.2 (2C), 62.3, 64.1 (2C), 64.2 (d, J ) 5.0 Hz), 66.3, 66.7, 67.0, 67.4,
68.1, 68.2, 71.0 (d, J ) 6.5 Hz), 71.4 (2C), 71.7 (3C), 71.9, 72.0, 72.9,
73.4, 73.6, 74.0, 74.1, 74.5 (2C), 75.7, 76.3, 76.6, 76.7, 77.0 (d, J )
5.4 Hz), 78.0, 78.2, 78.5 (d, J ) 5.6 Hz), 80.0, 82.6 (2C), 83.9, 84.0,
(2,3,5,6-tetra-O-Benzyl-â-D-galactofuranosyl)-(1 f 3)-2,4,6-tri-
O-benzyl-R-D-mannopyranosyl)-(1 f 2)-[(2,3,5,6-tetra-O-acetyl-â-
D-galactofuranosyl)-(1 f 3)]-4,6-di-O-benzyl-R-D-mannopyranosyl-
9
3418 J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006

Synthesis of T. cruzi LPPG Glycan
A R T I C L E S
96.3, 99.5, 101.9, 102.5, 102.8, 105.7, 106.0; 31P (decoupled, 121 MHz,
D2O, pH* ) 5.1), δ 22.4, 2.0; HRMS calcd. for C50H90N2O45P2: [M +
H]+ 1501.4370; Found: 1501.4343.
Swedish Foundation for Strategic Research (SSF). P.K. and
M.H. thank the Swedish Research Council (VR) for financial
support.
Acknowledgment. Dr. Christopher Jones, National Institute
for Biological Standards and Control, Herts UK, is thanked for
valuable information and comments on the manuscript. M.H.
is enrolled in the graduate school Forum Scientium and the
Biomimetic Materials Science program, supported by the
Supporting Information Available: 1H-, 13C-, and 31P NMR
spectra of all new compounds and complete ref 4. This material
is available free of charge via the Internet at http://pubs.acs.org.
JA057339B
9
J. AM. CHEM. SOC. VOL. 128, NO. 10, 2006 3419